Determining exon connectivity in complex mRNAs by nanopore sequencing

Bolisetty, Mohan; Rajadinakaran, Gopinath; Graveley, Brenton R.

doi:10.1186/s13059-015-0777-z

Cited by 143 publications

(123 citation statements)

References 15 publications

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“…To illustrate, Bolisetty et al [8] used the MinION to determine RNA splice variants and to detect isoforms for four genes in Drosophila . Among these is Dscam1 , the most complex alternatively spliced gene known in nature, with 18,612 possible isoforms ranging in length from 1806 bp to 1860 bp [8].…”

Section: Benefits Of Minion Compared To Other Next Generation Sequencmentioning

confidence: 99%

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The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community

et al. 2016

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Section: Benefits Of Minion Compared To Other Next Generation Sequencmentioning

confidence: 99%

“…Among these is Dscam1 , the most complex alternatively spliced gene known in nature, with 18,612 possible isoforms ranging in length from 1806 bp to 1860 bp [8]. They detected over 7000 isoforms for Dscam1 with >90% alignment identity.…”

Section: Benefits Of Minion Compared To Other Next Generation Sequencmentioning

confidence: 99%

The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community

et al. 2016

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“…A good agreement between the abundance of targeted cDNA amplicons sequenced with the ONT MinION and the Illumina MiSeq platforms, has been observed in another study10. Given that the platform can be used for accurate quantification of isoform expression abundance it is very important to be able to use as many sequenced reads as possible with the caveat of being of a lower quality.…”

Section: Discussionmentioning

confidence: 62%

Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

Oikonomopoulos

Wang

Djambazian

et al. 2016

Sci Rep

143

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To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

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“…Many of the events are reminiscent of wellstudied examples of high-entropy AS in other systems, such as the myriad of splice variants generated by tandem arrays of alternative exons in the Drosophila DSCAM gene 32 . In this classic example, high-entropy AS events overlap tandemly repeated immunoglobulin-like domains that function as interaction surfaces in neural circuit assembly 33 .…”

Section: Discussionmentioning

confidence: 99%

Whippet: an efficient method for the detection and quantification of alternative splicing reveals extensive transcriptomic complexity

Sterne-Weiler

Weatheritt

Best

et al. 2017

Preprint

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Alternative splicing (AS) is a widespread process underlying the generation of transcriptomic and proteomic diversity in metazoans. Major challenges in comprehensively detecting and quantifying patterns of AS are that RNA-seq datasets are expanding near exponentially, while existing analysis tools are computationally inefficient and ineffective at handling complex splicing patterns. Here, we describe Whippet, a method that rapidly, and with minimal hardware requirements, models and quantifies splicing events of any complexity without significant loss of accuracy.Using an entropic measure of splicing complexity, Whippet reveals that approximately 33% of human protein coding genes contain complex AS events that result in substantial expression of multiple splice isoforms. These events frequently affect tandem arrays of folded protein domains. Remarkably, high-entropy AS events are more prevalent in tumour relative to matched normal tissues, and these differences correlate with increased expression of proto-oncogenic splicing factors. Whippet thus affords the rapid and accurate analysis of AS events of any complexity, and as such will facilitate biomedical research.

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Determining exon connectivity in complex mRNAs by nanopore sequencing

Cited by 143 publications

References 15 publications

The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community

The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community

Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

Whippet: an efficient method for the detection and quantification of alternative splicing reveals extensive transcriptomic complexity

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